The purpose of this research is to identify, separate and characterize subpopulations of human periodontal fibroblasts. Fibroblasts are the major resident cells which inhabit the periodontal tissues. As such, they are crucial for maintaining the connective tissues which support and anchor the tooth. Little is known of their origins, synthesis of regulatory cytokines and growth factors in health and disease, and their importance in soft tissue regeneration. The research delineated in this proposal will help fill that gap. An emerging concept is that fibroblasts are dynamic cells which are not homogeneous, but instead consist of subsets of cells. Previous studies have demonstrated that fibroblasts from lung tissues can be separated into subsets on the basis of surface marker expression. Importantly, these fibroblast subsets possess distinct functions. Preliminary data obtained in support of this application, using fibroblasts derived from human gingiva and periodontal ligament (PDL), also indicate that they ar composed of subsets with distinct patterns of surface marker expression which will permit their separation into functional subpopulations. The hypothesis to be explored is that periodontal fibroblasts are not homogeneous, but consist of subsets which can be identified by discrete, heritable, cell surface markers. In this proposal the first aim is to establish optimal condiitons for isolating fibroblast lines from gingiva and PDL, and to identify cell surface markers most suited to the separation of gingival and PDL fibroblasts. Selected monoclonal antibodies and flow cytometry will be utilized to accomplish this goal. The second aim of this project is to separate gingival and PDL fibroblast subsets based on the expression of the selected surface markers. This goal will be accomplished using fluorescence activated cell sorting (FACS) to isolate various subsets so they can be cultured and cloned. Heritability and stability of surface marker expression of the established clones will be analyzed. By completing the goals outlined in this proposal it will be possible to study whether any unique functional differences exist between periodontal fibroblast subsets. The information derived from these experiments will lay the foundation for future studies of functional heterogeneity among fibroblast types. A significant potential outcome of this research is that new approaches for periodontal therapy, including regeneration, may be developed once a better understanding of subsets of periodontal fibroblasts is achieved.